Characterizing the Plant-Associated Symbionts, Rhodococcus and Agrobacterium

Symbioses are a spectrum of interactions between organisms living in closeassociation. These intimate interactions range from mutualism, in which bothorganisms benefit, to parasitism, where one organism benefits at the expense ofthe other. Horizontal gene transfer is the acquisition of genes independent ofvertical transmission and demonstrably promotes the transition of bacteria fromfree-living to symbiotic. The horizontal acquisition of plasmids allows members ofthe Agrobacterium and Rhodococcus genera to cause disease to plants, many ofwhich are important for the ornamental plant industry. Therefore, the accurate andrapid diagnosis of these pathogens is critical for management, and failure toproperly diagnose or respond can result in severe economic losses. Indeed, somediagnostic methods have the potential to be misleading and assign nonpathogenicRhodococcus as the causative agents of disease. I test the hypothesis thathorizontal gene transfer can elicit the transition of Rhodococcus between beneficialand pathogenic states.I demonstrate that most Rhodococcus isolates are beneficial and promotechanges to the root system, such as those that are frequently associated with plantgrowth promoting bacteria. I further demonstrate that beneficial Rhodococcusstrains transition to pathogens upon the acquisition of a virulence plasmid. Theseare virulent on plants and can cause disease symptoms to both roots and aerialportions of plants. Lastly, I develop reagents for use in a sensitive and specificnovel diagnostic test that accelerates identification of pathogenic strains ofRhodococcus and Agrobacterium. This work highlights the fluidity in the evolutionof plant-associated bacteria and how transitions in symbiotic state can confounddiagnosis.Keywords: Rhodococcus, Symbiosis, Detection, Agrobacteriu

Development of a new molecular diagnostics tool for Agrobacterium tumefaciens

Agrobacterium tumefaciens causes the economically important plant disease, crown gall. These galls are the result of abnormal growth that manifests itself on either the root or the stem and can be devastating for the ornamental plant industry. Introduction of A. tumefaciens to a nursery can be highly detrimental and thus early detection of the pathogen is crucial in order to limit its spread. Current methods of pathogen identification such as polymerase chain reaction (PCR) are costly, require specialized equipment, and can take a significant amount of time. By optimizing an isothermal recombinase polymerase amplification (RPA) and lateral flow analysis, the rapid and sensitive detection of A. tumefaciens can be done in the production setting, and does not require specialized equipment. This is possible because recombinase proteins are capable of separating double stranded DNA without the need for high temperatures. Once the RPA reaction is complete, a probe is used to bind to DNA fragments amplified from a region of the A. tumefaciens plasmid. Amplification is visualized using lateral flow analysis, aka, a dipstick, eliminating the need for gel electrophoresis. I have developed a primer set and a probe for RPA detection of phytopathogenic A. tumefaciens that is specific, sensitive, and has been optimized for reaction time, temperature, and is accurate at low DNA concentrations. I selected a gene, virD2, which is present in all known pathogenic strains of A. tumefaciens as the target for RPA. The primer set has been optimized for high specificity, yet can identify various A. tumefaciens strains. The assay was tested against phytopathogenic and non-pathogenic bacteria as well as plant samples and was shown to be specific to virD2-containing A. tumefaciens. I have also determined the ideal temperature and time for an RPA is 37℃ for 30 minutes. By optimizing these conditions, the presence or absence of A. tumefaciens can be accurately detected by applying the RPA product to the lateral flow analysis strip. If present, a band will appear on the strip in less than one minute. Development of this assay will provide growers with a fast, inexpensive, and accurate method of detecting pathogenic A. tumefaciens

Coral Identification Guide NAFO Area

Accurate reporting of benthic corals is increasingly important for mapping distributions and for the continued development of sustainable fisheries under the ecosystem approach. This coral identification guide is intended to help those on-board commercial and research fishing vessels to identify and record the various species of coral likely to be commonly encountered in fishing trawls. The guide is clear and simple to use, and will provide names to the majority of these beautiful bottomdwelling animals. The photographs are typically of caught specimens taken on the deck, as this gives the best picture of what is actually seen. Sadly, we rarely personally see corals in their natural habitat, except by looking at films and photos taken by deep underwater cameras

Competency-Based Time-Variable Anesthesiology Residency Training: Identification of Problems and Solutions

BACKGROUND: Global medical education is gradually moving toward more comprehensive implementations of a competency-based education (CBE) model. Elimination of standard time-based training and adoption of time-variable training (competency-based time-variable training [CB-TVT]) is one of the final stages of implementation of CBE. While CB-TVT has been implemented in some programs outside the United States, residency programs in the United States are still exploring this approach to training. The Accreditation Council for Graduate Medical Education (ACGME) and the American Board of Medical Specialties (ABMS) are encouraging member boards and residency review committees to consider innovative ways programs could implement CB-TVT. The goals of this study were to (1) identify potential problems with the implementation of CB-TVT in anesthesiology residency training, (2) rank the importance of the problems and the perceived difficulty of solving them, and (3) develop proposed solutions to the identified problems. METHODS: Study participants were recruited from key stakeholder groups in anesthesiology education, including current or former program directors, department chairs, residents, fellows, American Board of Anesthesiology (ABA) board members, ACGME residency review committee members or ACGME leaders, designated institutional officials, residency program coordinators, clinical operations directors, and leaders of large anesthesiology community practice groups. This study was conducted in 2 phases. In phase 1, survey questionnaires were iteratively distributed to participants to identify problems with the implementation of CB-TVT. Participants were also asked to rank the perceived importance and difficulty of each problem and to identify relevant stakeholder groups that would be responsible for solving each problem. In phase 2, surveys focused on identifying potential solutions for problems identified in phase 1. RESULTS: A total of 36 stakeholders identified 39 potential problems, grouped into 7 major categories, with the implementation of CB-TVT in anesthesiology residency training. Of the 39 problems, 19 (48.7%) were marked as important or very important on a 5-point scale and 12 of 19 (63.2%) of the important problems were marked as difficult or very difficult to solve on a 5-point scale. Stakeholders proposed 165 total solutions to the identified problems. CONCLUSIONS: CB-TVT is a promising educational model for anesthesiology residency, which potentially results in learner flexibility, individualization of curricula, and utilization of competencies to determine learner advancement. Because of the potential problems with the implementation of CB-TVT, it is important for future pilot implementations of CB-TVT to document realized problems, efficacy of solutions, and effects on educational outcomes to justify the burden of implementing CB-TVT

Effects of alterations in positron emission tomography imaging parameters on radiomics features.

Radiomics studies require large patient cohorts, which often include patients imaged using different imaging protocols. We aimed to determine the impact of variability in imaging protocol parameters and interscanner variability using a phantom that produced feature values similar to those of patients. Positron emission tomography (PET) scans of a Hoffman brain phantom were acquired on GE Discovery 710, Siemens mCT, and Philips Vereos scanners. A standard-protocol scan was acquired on each machine, and then each parameter that could be changed was altered individually. The phantom was contoured with 10 regions of interest (ROIs). Values for 45 features with 2 different preprocessing techniques were extracted for each image. To determine the impact of each parameter on the reliability of each radiomics feature, the intraclass correlation coefficient (ICC) was calculated with the ROIs as the subjects and the parameter values as the raters. For interscanner comparisons, we compared the standard deviation of each radiomics feature value from the standard-protocol images to the standard deviation of the same radiomics feature from PET scans of 224 patients with non-small cell lung cancer. When the pixel size was resampled prior to feature extraction, all features had good reliability (ICC > 0.75) for the field of view and matrix size. The time per bed position had excellent reliability (ICC > 0.9) on all features. When the filter cutoff was restricted to values below 6 mm, all features had good reliability. Similarly, when subsets and iterations were restricted to reasonable values used in clinics, almost all features had good reliability. The average ratio of the standard deviation of features on the phantom scans to that of the NSCLC patient scans was 0.73 using fixed-bin-width preprocessing and 0.92 using 64-level preprocessing. Most radiomics feature values had at least good reliability when imaging protocol parameters were within clinically used ranges. However, interscanner variability was about equal to interpatient variability; therefore, caution must be used when combining patients scanned on equipment from different vendors in radiomics data sets